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. 2003 Feb;62(2):100–107. doi: 10.1136/ard.62.2.100

Angiopoietin-1 is expressed in the synovium of patients with rheumatoid arthritis and is induced by tumour necrosis factor α

E Gravallese 1, A Pettit 1, R Lee 1, R Madore 1, C Manning 1, A Tsay 1, J Gaspar 1, M Goldring 1, S Goldring 1, P Oettgen 1
PMCID: PMC1754433  PMID: 12525377

Abstract

Objectives: To examine the potential role of the angiogenic growth factor angiopoietin-1 (Ang-1) in inflammatory arthritis.

Methods: Eighteen synovial tissue samples were obtained from 17 patients with a clinical diagnosis of rheumatoid arthritis (RA) and compared with six synovial tissue samples from six patients with osteoarthritis (OA). Ang-1 expression in synovial tissues was determined by immunohistochemistry and in situ hybridisation. Ang-1 mRNA and protein expression were also examined by northern blot analysis and enzyme linked immunosorbent assay (ELISA) in cultured synovial fibroblasts and human umbilical vein endothelial cells (HUVECs) before and after treatment with tumour necrosis factor (TNF)α.

Results: Ang-1 protein expression was detected by immunohistochemistry in 16/18 RA synovial tissue samples. Ang-1 protein was frequently observed in the synovial lining layer and in cells within the sublining synovial tissue, in both perivascular areas and in areas remote from vessels. In contrast, Ang-1 was only weakly detected in these sites in OA samples. Ang-1 mRNA and protein were also expressed in cultured synovial fibroblasts derived from patients with RA. In addition, induction of Ang-1 mRNA and protein was observed by northern blot analysis and ELISA after stimulation of RA synovial fibroblasts, but not HUVECs, with the proinflammatory cytokine TNFα.

Conclusions: Ang-1 mRNA and protein are expressed in the synovium of patients with RA, and are up regulated in synovial fibroblasts by TNFα. Ang-1 may therefore be an important regulator of angiogenesis in inflammatory arthritis.

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Figure 1.

Figure 1

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Immunohistochemical staining of Ang-1 in RA and OA synovial tissue. (A) Ang-1 protein is demonstrated in RA synovial tissue in cells in the synovial lining layer, in some endothelial cells, and in cells within subsynovial tissue. (B) Isotype matched control antibody staining, serial section. (C) Cells in perivascular areas of RA synovial tissue expressing Ang-1 protein. (D) Cells in RA subsynovial tissues expressing Ang-1 protein. (E and F) Weak Ang-1 expression was seen in OA synovial tissue samples in the lining layer, and occasionally in cells within subsynovial tissues. (G and H) Serial sections of RA synovial tissue stained for CD68 (G) and Ang-1 (H). (I) Isotype matched control antibody staining for CD68 in a serial section (magnification x150). Original magnification in (A)–(F) is x50 and in (G) and H x150.

Figure 2.

Figure 2

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In situ hybridisation for Ang-1 and Ang-2 in rheumatoid synovium. Ang-1 in situ hybridisation in synovial tissue samples from patients with RA using the Ang-1 antisense (A, C–F) and sense (B) probes, demonstrating representative areas of mRNA expression for Ang-1. Probe hybridisation is noted in isolated cells within the subsynovial tissues (A), and within cells in the wall of a subsynovial arteriole (C, arrow), shown at higher magnification (D). (E and F) Low and high power views of Ang-1 hybridisation within cells of the synovial lining layer seen in two samples. In situ hybridisation using the Ang-2 antisense probe demonstrating probe hybridisation within a subsynovial vessel at low (G, arrow) and high power magnification (H). Original magnification in (A), (B), and (E–G) is x50 and in (C), (D), and (H) x150.

Figure 3.

Figure 3

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Northern blot analysis of Ang-1 and Ang-2 in cultured synovial fibroblasts and human umbilical endothelial cells. (A) Induction of Ang-1 mRNA expression in cultured human synovial fibroblasts in response to TNFα (10 ng/ml) at baseline (lane 1) and 1, 2, 4, 6, 12, and 24 hours after stimulation with TNFα (lanes 2–7). (B) Repression of Ang-1 mRNA expression in cultured human umbilical vein endothelial cells in response to IL1ß (10 ng/ml) and TNFα (10 ng/ml) at baseline (lanes 1, 6) and 2, 4, 6, and 24 hours after stimulation with IL1ß (lanes 2–5) and TNFα (lanes 7–10). (C) Repression of Ang-2 mRNA expression in cultured human umbilical vein endothelial cells in response to IL1ß (10 ng/ml) and TNFα (10 ng/ml) at baseline (lanes 1, 6) and 2, 4, 6, and 24 hours after stimulation with IL1ß (lanes 2–5) and TNFα (lanes 7–10).

Figure 4.

Figure 4

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ELISA analysis of Ang-1 protein in culture media of RA synovial fibroblasts. Ang-1 protein was quantified in the culture media of synovial fibroblasts stimulated with TNFα (10 ng/ml) at 0, 4, 18, 24, and 30 hours in this representative experiment. Experiments were performed in triplicate and are expressed as means (standard deviation).

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